Provocative question (PQ) 5 challenges us to determine the mechanism whereby 'drugs commonly used for other indications, such as anti-inflammatory drugs (NSAIDs), can protect against cancer incidence and mortality'. Strong preliminary data lead us to propose that for certain chemical entities the chemopreventive and/or anti-tumor activity is attributable to interaction of the R-enantiomer of select NSAIDs with novel cancer- relevant targets. There are many examples of stereoselective differences in drug activity. The R-forms of non- steroidal anti-inflammatory drugs (NSAIDs) are essentially inactive against cyclooxygenases and there is mounting evidence that R-enantiomers are distinct chemical entities with independent pharmacologic activities. We find that the R-enantiomers of naproxen and ketorolac inhibit the small GTPases Rac1 and Cdc42. More than 20 other NSAIDs were inactive against these proteins, suggesting novel target selectivity by R-naproxen and R-ketorolac. Rac1 and Cdc42 regulate cytoskeletal dynamics in addition to other functions, and have been recognized as attractive cancer therapeutic targets although no specific inhibitors are currently in clinical trials. In keeping with the known functions of Rac1/Cdc42 in regulating actin-based functions, we find enantiomer- selective inhibition of ovarian tumor cell migration and adhesion. Furthermore, R-naproxen, but not S-naproxen or the structurally-related 6-methoxy naphthalene acetic acid (6-MNA), reduced implantation of ovarian tumors by ~75% in an intraperitoneal xenograft model. The objective of this application is to define the mechanism of action for the observed biologic activities of R-naproxen and R-ketorolac. This will provide the foundations for re-consideration of potential anti-tumor activities of other NSAID R-enantiomers based on interaction with novel targets. We hypothesize that R-ketorolac and R-naproxen inhibit Rac1 and/or Cdc42 and associated cellular responses through a novel mechanism based on drug binding to the GDP-bound (inactive) form of the enzymes. We will test this hypothesis by using biochemical and cellular approaches coupled with structure- activity analyses, we will test whether there is R-enantiomer-selective interaction with the GDP-bound forms of the GTPases as predicted by cheminformatics, leading to blockade of GTP binding, enzyme activation and downstream cellular responses. The work is significant because the novel pharmacologic and tumor-relevant functional activities of NSAID R-enantiomers have not been previously described and successful completion of the project will offer new mechanistic insights into the anti-cancer benefit of NSAIDS. The studies will also yield additional and critical information on the benefits of targeting Rac1 and Cdc42 in ovarian and other cancers. PUBLIC HEALTH RELEVANCE: The benefits of non-steroidal anti-inflammatory drugs (NSAIDs) as general chemopreventive and anti-cancer agents remains unresolved due to mixed results from large population-based studies. Inconsistencies in the published findings may be due to previously unappreciated activities of certain NSAIDs and our team, encompassed by the University of New Mexico Center for Molecular Discovery and the University of Kansas Specialized Chemistry Center, has discovered that two NSAID derivatives (both FDA approved, off-patent drugs) are selective for new cancer-relevant targets with known roles in cancer growth, migration and metastasis, namely Rho-family GTPases. By defining the mechanism by which these NSAIDS inactivate the Rho-family GTPases, we expect to provide evidence to support potential new clinical applications for specific NSAIDS in cancer prevention or therapeutics.